Claims have been made recently that glyphosate-resistant (GR) crops sometimes have mineral deficiencies and increased plant disease. This review evaluates the literature that is germane to these claims. Our conclusions are: (1) although there is conflicting literature on the effects of glyphosate on mineral nutrition on GR crops, most of the literature indicates that mineral nutrition in GR crops is not affected by either the GR trait or by application of glyphosate; (2) most of the available data support the view that neither the GR transgenes nor glyphosate use in GR crops increases crop disease; and (3) yield data on GR crops do not support the hypotheses that there are substantive mineral nutrition or disease problems that are specific to GR crops.

Since the herbicide glyphosate (N-(phosphonomethyl)glycine)
was commercialized in 1974, it has become the most widely used herbicide
in the world, due largely to the wide scale adoption of transgenic,
glyphosate-resistant (GR) crops after their introduction in 1996 (Figure 1). In GR crops, this relatively high use rate herbicide
(commonly 0.5 to 2.0 kg/ha/application) is often used multiple times
in a growing season. Use of other herbicides declined steadily, while
glyphosate use increased in the three major GR crops (Figure 2). The increasing incidence of evolved, GR weeds,2 as well as weed shifts to naturally glyphosate-tolerant
weed species,3 has resulted in increased
use rates and numbers of applications of glyphosate, as well as other
herbicides, per growing season in GR crops. Since its introduction,
glyphosate has been considered a toxicologically and environmentally
safe pesticide, due to its low mammalian toxicity, relatively short
environmental half-life, and extremely low activity in soil due to
its binding to soil minerals (reviewed by Duke et al.4). Furthermore, only green plants, some fungi, and a limited
number of microorganisms have the target site, 5-enolpyruvylshikimic
acid-3-phosphate synthase (EPSPS), of the herbicide. EPSPS is an enzyme
required for synthesis of the essential aromatic amino acids phenylalanine,
tyrosine, and tryptophan.

Glyphosate has several desirable properties that
have contributed
to its widespread use.5 Glyphosate is a
nonselective herbicide, that is, it can kill all plant species, although
there is variation between species with regard to levels of natural
tolerance. Glyphosate has little or no herbicidal activity in soil
and, thus, is used only with foliar spray applications. Due to crop
sensitivity, its use was limited in crop production prior to the introduction
of GR crops, after which its use greatly expanded with the widespread
adoption of these crops worldwide.

During its several decades
of use over vast areas, no significant
adverse secondary effects of the herbicide have been established,
other than the intense selection pressure that has resulted in the
evolution of GR weeds. In fact, its use in GR crops has been associated
with several environmental benefits.6,7 The topic of
evolution of GR weeds has been dealt with in detail in many research
papers and reviews.8,9

Several papers have been
published recently that conclude that
glyphosate adversely affects mineral nutrition in GR crops, leading
to several adverse effects, including increased plant disease.10−20 Others21 have indicated that GR crops
are more susceptible to plant diseases due to other mechanisms. This
review addresses these concerns in the context of the available literature
on glyphosate (ca. 8000 peer-reviewed papers according to SciFinder).

Glyphosate in Soil: Bioavailabiltiy, Degradation, and Persistence

In order to understand possible effects of glyphosate on mineral
nutrition of plants, it is necessary to understand the processes that
affect glyphosate in soil. It is also necessary to understand how
glyphosate interacts with minerals in soil and with soil microorganisms.

Sorption/Bioavailability

Once glyphosate interacts
with soil, whether applied directly to the soil surface, exuded from
a plant root, or released from decomposing plant tissue, it is subject
to various processes that control its environmental behavior and fate,
including retention (sorption–desorption), transport, and degradation.
Of these processes, sorption is arguably the most important as it
controls the availability for degradation, plant uptake, and offsite
transport. Sorption of glyphosate to soil has been extensively reviewed.22−24 Because glyphosate is a small polyprotic molecule (pKa1 = 2.27, pKa2 = 5.58, pKa3 = 10.2522) with three polar functional groups and can
be sorbed on minerals and organic matter, its sorption on soil as
a whole is generally much greater compared to other pesticides, which
are larger molecules with fewer functional groups and are primarily
sorbed onto organic matter.

Glyphosate is primarily sorbed on
variable-charge surfaces such as iron and aluminum oxides, aluminum
silicates (allophone and imogolite), and goethite (α-FeOOH),
and to a lesser extent on the Fe-oxide coatings of permanent charge
minerals (illite, smectite, and vermiculite) and organic matter. The
primary mechanisms responsible for sorption are ligand exchange and
complex formation with mineral oxide surfaces. The magnitude of sorption
increases with increased surface area of the minerals and decreased
pH. The sorption onto the sorbent surface is fast initially for most
of the glyphosate added to soil, which is then followed by slower
sorption. Further details of these processes can be found in the above
cited reviews and the references cited therein.

The magnitude
of sorption has traditionally been characterized
as the ratio of glyphosate bound to soil to that in solution for a
single concentration (Kd) or at multiple
concentrations (Kf and 1/n values from the Freundlich sorption isotherm). Sorption coefficients
are often expressed on a soil organic carbon basis (Koc, Kfoc) to normalize values
between different soils. Glyphosate is strongly bound to soil. For
instance, regardless of soil properties in a cultivated prairie, glyphosate
mean Kd was 108 to133 L kg–1 (Koc = 10 900–14 900
L kg–1), depending on landscape position, and these
values were 100× greater than those for the commonly used herbicide
2,4-D.25 In a study of 20 different soils, Kd ranged from 41 to 303 L kg–1 with a median value of 97 L kg–1.26 In column leaching experiments, coarse textured soils retained
most all (85–95%) of the glyphosate applied despite the fact
that higher than agronomic rates, 7.4–14.8 mg kg–1, of glyphosate were used.27

After
glyphosate is sorbed, it is not readily desorbed. Desorption
is inversely related to adsorption, being small when sorption is great.28 Depending on soil type, glyphosate is weakly
desorbed, that is, 5–24% of initially sorbed glyphosate.29 The strong adsorption of glyphosate to most
soils, and its low desorbability, leaves little glyphosate in soil
solution available for microbial degradation, interaction with trace
metal cations, plant uptake, or offsite transport.

Sorbed glyphosate
has been postulated to be released into soil
solution upon addition of phosphate (PO43-), which can compete with glyphosate for sorption sites on soil.24 However, it appears there is only limited competition
for sorption sites between glyphosate and PO43-, even when much higher than agronomic rates of glyphosate are applied.27 For soils, competitive sorption studies between
glyphosate and PO43- showed that displacement
of glyphosate by PO43- was related to
the amount of clays, CEC, and pH, but glyphosate was not easily displaced
by PO43- from the clays.30 Even when sorption competition has been shown to occur,
glyphosate still remains strongly sorbed. For example, increasing
extractable P by a factor of 10 in soil, only decreased the sorption
coefficient Kf from 215 to 106 L kg–1 in loamy sand and 154 to 84 L kg–1 in coarse sand soils.31 As a result,
solution concentration of glyphosate does not appreciably increase
upon the addition of PO43- at environmentally
relevant P concentrations. Also, the competition between glyphosate
and PO43- (if it occurs) appears to be
temporary; the same amounts of glyphosate and PO43- were sorbed after ∼7 days whether the compounds are present
alone or together.32 Therefore, it seems
likely that glyphosate and PO43- are
specifically sorbed on to common as well as specific sites on various
soil components.

Glyphosate can form chelates or complexes with
micronutrient metal
ions in solution. At physiologically relevant pH levels, and pH levels
of most soils, Cu and Zn ions in solution can be relatively strongly
complexed with glyphosate, whereas Fe, Ca, Mg, and Mn are complexed
to lesser degrees.22,33 Because of the ability of glyphosate
to complex metal ions, glyphosate has been postulated to affect plant
uptake of trace nutrients such as Mn2+ or Zn2+. For plants grown in hydroponic solutions, mixed results for glyphosate
effects on plants have been shown. In contrast, Andrade and Rosolem34 reported that glyphosate did not affect Mn absorption
and transport in GR soybean plants in the field. This topic is discussed
in more detail in the section below on the effects of glyphosate on
mineral nutrition of plants.

It is difficult to extrapolate
hydroponic studies to field situations
where there are numerous cations at varying concentrations that can
form complexes with glyphosate, and where soluble metal-glyphosate
complexes are subject to sorption processes on soil, as are glyphosate
and metal ions. For example, the presence of Zn increased glyphosate
sorption on two soils as a result of decreased solution pH resulting
from Zn2+ exchanging with H+ on the soil surface.35 In bioassay experiments using tomato plants
and white spruce seedlings, soils containing saturated solutions of
glyphosate-metal complexes had little or no effect on
the plants.33 In a recent study of micronutrient
accumulation in soybean grown using standard agronomic practices in
Indiana, results showed that while there were differences in accumulation
of micronutrients between cultivars, there was no consistent effect
due to glyphosate treatment.36

Micronutrient
metal concentrations in soil solutions can vary spatially
and temporally. For example, in five woodland sites, the mean soil
solution concentrations of total Mn and Zn were 69 and 1.8 μmol
L–1, respectively, while in grassland sites, total
Mn concentrations varied by a factor of 6 in a clay soil and a factor
of 2 in a sandy soil.37 In a soil toposequence
under natural vegetation, total Mn concentration in soil solution
varied by a factor of 900, depending on topographic soil position.38 In addition, the metal cations in soil solution
are not necessarily free ions; they can form complexes with dissolved
soil organic matter and other ligands. For example, in a study of
15 agricultural soils, total soil solution concentrations of Cu ranged
from 0.023 to 1.03 μmol L–1, with free Cu2+ comprising 7–73% of total dissolved Cu.39 In the same soils, total Zn solution concentration
ranged from 0.4 to 43 μmol L–1, with free
Zn2+ comprising 47–99% of total dissolved Zn. Glyphosate
can only compete with other soil ligands and sorbent surfaces for
free metal ion activity, with most glyphosate being adsorbed by the
soil rather than remaining in the soil solution where it can complex
with metal ions.

In spite of the wide range of micronutrient
cation concentrations
in soil solutions, their concentrations are much greater than would
be found for glyphosate in soil solutions. Using a glyphosate application
rate of 1 kg ha–1 (soil concentration = 0.75 μg
g–1, assuming incorporation to a depth of 10 cm
and a soil bulk density of 1.33), and an average soil Kd = 100, the amount of glyphosate in soil solution would
be 0.044 μmol L–1, which is much smaller than
typical Mn2+, Zn2+, Cu2+, and Fe3+ concentrations found in soil solutions from agricultural
soils. Under agricultural production, concentrations of, Mn2+, Zn2+, Cu2+, and Fe3+ in soil solutions
of Holtville (Typic Torrifluvent) and Altamont (Typic Chromoxerert)
soils would be on average 480×, 220×, 80×, and 310×
greater than the glyphosate in solution, respectively.40 Therefore, free cation concentrations, such as Mn2+, would not be reduced appreciably by glyphosate addition to soil,
even at the highest recommended application rates and assuming all
the glyphosate in solution formed a chelate with Mn. Furthermore,
glyphosate degrades rapidly (see degradation section below), whereas
although micronutrient concentrations in soil can fluctuate temporally
during the year, micronutrients do not degrade.

Glyphosate Degradation, Persistence, and Leaching

Biological Degradation Pathway

The degradation of glyphosate
in soil has been extensively documented.24,41 The primary
degradation pathway is the cleavage of glyphosate by glyphosate oxidoreductase
to glyoxylate and AMPA (aminomethylphosphonic acid) (Figure 3), with the latter subsequently degraded to methylamine
and inorganic phosphate by C–P lyase enzymes. Both glyoxylate
and methylamine can support the growth of microorganisms. Alternatively,
the transformation of glyphosate to AMPA and glyoxylate can also be
performed by glycine oxidase.42

A second degradation pathway is the cleavage of
inorganic phosphate
from glyphosate by C–P lyase producing the sarcosine metabolite
(Figure 3). Sarcosine is further degraded into
formaldehyde and glycine, which are utilized by a wide variety of
soil microorganisms. Soil microorganisms utilizing the sarcosine pathway
have been isolated and characterized,43 including members of the soybean root symbionts, the Rhizobiaceae.44 Soil fungi also degrade glyphosate, and AMPA
was reported as a metabolite.45 Since some
microorganisms are sensitive to glyphosate (see this review), the
degradation of glyphosate in situ represents the activity of the glyphosate-degrading
microbial community modulated by relative resistance or sensitivity
to the herbicide.

14C-Glyphosate Fate Studies

Several studies
have utilized 14C-labeled glyphosate to examine the fate
of glyphosate in soil. These methods are useful because they provide
an integrated assessment of glyphosate degradation by measurement
of 14CO2 production (mineralization) and the
incorporation of glyphosate and glyphosate degradation products into
soil organic matter and biota (bound 14C residue). The 14CO2 produced from microbial degradation after
glyphosate addition to soil is variable, ranging from ≤10%
in 332 d,46 to <10–40% in 96
d,47 to 50–70% in 35 d,48 to >70% in 140 d,49 and depends on soil type and 14C-label location (phosphomethyl-14C versus aminomethyl-14C). The production of 14CO2 begins after addition of glyphosate to soil
without a lag period, showing that microorganisms with the capacity
to degrade glyphosate are present in most all soils. The studies conducted
with 14C label in the phosphonomethyl carbon show degradation
of the AMPA metabolite. The variability in the amount of 14CO2 produced from glyphosate degradation in soil is likely
due to the variability in the population of glyphosate-degrading microorganisms
present in soil (the glyphosate-degrading microbial community in soil
has not been fully characterized) and their biological activity and
to competing sorption and binding processes.

In addition to
the direct application of glyphosate to soil, any glyphosate remaining
in GR crop residues will be released to the soil as those crop residues
degrade.50 At 35 days after treatment,
glyphosate residues within corn leaves mineralized more slowly (61%)
than glyphosate applied directly to soil (77%).51

The use of GR crops allows for multiple applications
(commonly
two or three) of glyphosate within a field each growing season. 14C-labeled glyphosate has also been used to determine whether
repeated applications of glyphosate can affect glyphosate degradation
in soil. Repeated applications reduced the rate of glyphosate mineralization
by 28% from the initial application to the fifth application in a
10-wk period (Figure 4). However, there was
no difference in the rate of 14C-glyphosate mineralization
between one, two, three, or four glyphosate applications.52 While the rate of mineralization was decreased
after the fifth application relative to the first, it was not reduced
relative to the second, third or fourth application. In a similar
experiment, Andréa et al.53 found
that the initial, immediately mineralizable glyphosate decreased after
sequential applications as compared to the initial application. However,
after the initial mineralization, the rate of mineralization was the
same. For instance, the differences in the total amount of 14CO2 detected 8 wks after treatment and the immediately
mineralizable glyphosate were 16 and 19% after 1 and 4 sequential
applications, respectively. Weaver et al.54 reported greater mineralization (% of applied) from glyphosate added
at 1× the field rate (47 mg glyphosate/kg soil, assuming 0.84
kg/ha application distributed in the surface 2 mm of soil) than at
the 3× rate of application. However, on a mass basis more glyphosate
was degraded in the soil receiving the 3× application. These
studies show that repeated glyphosate applications are unlikely to
severely reduce the ability of the soil microbial community to metabolize
glyphosate. Broad spectrum measures of microbial activity (respiration
and enzyme activities) and community structure show inconsistent or
no response to glyphosate use (see Effects on Rhizosphere
Populations and Community Structure section).

In addition to 14C-glyphosate being
mineralized, a portion
of the 14C-labeled glyphosate or its metabolites is converted
to microbial biomass and some remains unextractable from soil. The
amount of bound 14C-residue formed depends upon the molecular
location of the 14C-label, the soil interaction, and the
extraction methods used. Mamy et al.49 compared
the bound residue formed from several pesticides and found that glyphosate-bound
residues generally accounted for less than 20% of the initial 14C added, equivalent to trifluralin, but less than those formed
after application of the 14C-labeled herbicides metazachlor,
metamitron, and sulcotrione. Weaver et al.54 applied glyphosate equivalent to 1× or 3× recommended
field rates, and less than 10% of the 14C was present in
bound residues at 42 days after application. After four53 or five52 sequential
applications at 14-day intervals, less than 30% of the 14C from applied glyphosate was present as bound residue in the soil,
and there was no difference in bound residue formation after the first
or the fifth application. However, greater accumulation of bound residues
would occur from multiple applications than a single application.
Simonsen et al.55 assessed the bioavailability
of bound residues formed after a single glyphosate application to
plants by incubating 14C-glyphosate in soil for 6.5 months,
followed by planting canola or barley. Glyphosate was not detectable
in soil at planting, and after 41 days of plant growth only 0.006%
of the applied 14C was detected in the plants.

Persistence and Leaching in Field and Lysimeter Studies

Field dissipation rates of glyphosate are affected by soil properties,
method of application, and environmental conditions such as moisture
and temperature, and therefore are extremely variable.24 Field studies often result in longer estimated times to
50% dissipation (DT50) as compared to laboratory studies,
which are generally conducted under optimum conditions for degradation.
In one field study, under the application and weather conditions that
prevailed (which resulted in water-saturated soils), no glyphosate
was detected 24 h after treatment, with a trace amount detected in
one replicate soil sample after 1 year.56 Warm temperatures at the time of and in the season before the application
are thought to explain the fast degradation rate in the water-saturated
soil samples. In a comparison among glyphosate treatments between
a forest floor and mineral soil, the DT50 times for glyphosate
were 12 and 10 days for the forest floor matrix and mineral soil,
respectively.57 Simonsen et al.55 measured a 9-day DT50 for glyphosate
and 32-day DT50 for AMPA in soil. In an agronomic field
study, glyphosate dissipation in the surface soil was rapid (DT50 = 25 days) and only 10% of applied chemical was present
at 34 days after application.58 In another
field study, glyphosate had an estimated DT50 of 45–60
days, with total soil residues of glyphosate accounting for 6–18%
of initial chemical at 360 days after application.59 Bergström et al.60 reported
a 110 to 151 day DT50 for glyphosate in a clay soil and
attributed the long persistence to adsorption (Kf > 118).

As a result of strong sorption and slow
desorption,
some glyphosate residues tend to stay in the surface soils through
the growing season. For example, of the initial amount of glyphosate
added to a clay soil, 59% (glyphosate + AMPA residues) remained primarily
in the surface soil 748 days after application, despite large amounts
of precipitation after application.60 Also,
only 0.009 and 0.019% of the initial amount of glyphosate added leached
from the sand and clay soils, respectively, during the study period.
No leaching of AMPA occurred in the sand, whereas 0.03 g ha–1 leached in the clay soil.

Longer glyphosate persistence in
colder climates has been observed.
The DT50 time of glyphosate was generally <5 months
in Swedish railway embankments.61 In Northern
climates with seasonally frozen soils, field studies have shown clear
overwinter persistence for glyphosate. After glyphosate applications
in June and July, about 10–20% of applied glyphosate was detected
in the subsequent June in two field sites, demonstrating that the
time for 90% (DT90) dissipation of glyphosate was about
11 months.62 Similar overwinter persistence
was observed in agricultural fields in southeast Finland.63 Under warmer climates, glyphosate did not persist
past the growing season, even after 15 consecutive annual applications.64

After either pre-emergence or postemergence
applications of glyphosate,
the distribution of residues is nonuniform in soils and is more concentrated
near the surface of the soil. Almost two years after application to
a tilled soil in an outdoor lysimeter, glyphosate accounted for 1%
and AMPA for 19% of the applied glyphosate in the 0–10 cm depth
increment.65 In the 10–20 cm depth
increment, glyphosate was not detectable, and AMPA accounted for 5%
of the applied herbicide. Deep (>1 m) leaching of glyphosate has
been
observed, but concentrations in leachate were <0.07 μg L–1.23,60,66 This was attributed to movement in macropore flow, rather than leaching
through the bulk soil. Deep movement of glyphosate might be also expected
via translocation of the herbicide sprayed on to foliage of crops
and weeds to their roots, particularly resulting from glyphosate applications
later in the growing season.

Multiple applications of glyphosate
in GR-cropping systems would
(1) increase the risk of carryover, especially in regions where soils
are seasonally frozen for extended periods and (2) increase the risk
of leaching to tile drains or groundwater. Multiple applications of
glyphosate increase the time that bioavailable glyphosate is present
in soil. Also, plant interception of glyphosate in the field may lead
to a delayed release of glyphosate into the soil following foliage
decomposition. The degree of metabolic degradation of glyphosate in
plants41 would influence how much glyphosate
is released into soil by degradation of plant material. Such delayed
releases would increase DT50 times. Both of these processes
have been investigated in laboratory experiments, but corresponding
field studies are not yet available. Any increased persistence is
potentially important in cropping systems where glyphosate-sensitive
(GS) crops closely follow the GR-crop. The risks associated with planting
highly sensitive crops shortly after (<3 days) glyphosate applications
were known long before the advent of GR-crops.67,68

Glyphosate and Mineral Nutrition of Plants

During the
course of their action on susceptible plants, herbicides
eventually affect almost every physiological and biochemical process,
including mineral nutrition. Thus, glyphosate would be expected to
affect mineral nutrition of GS plants at herbicidal doses, but not
of GR plants at the same doses. Recent reports of mineral deficiencies
in GR crops after glyphosate application were linked with claims of
increased susceptibility to plant diseases.11,15,16,6 There are conflicting
papers on the effects of glyphosate on mineral nutrition on GR crops.
This is a complex topic that, for clarity, we have separated into
the aspects listed below.

Phytotoxicity of Metal Chelators

Many natural metal
chelators such as organic and amino acids are found in plant cytoplasm
and xylem and phloem fluids. Citrate is an important chelator of Fe
in xylem fluid, while some amino acids chelate metals in the cytoplasm
where the higher pH favors chelation by amino acids compared to organic
acids.70 Synthetic chelators have been
used in agriculture since 1950 to supply Fe or Zn to plants, and more
recently to induce phytoextraction of soil metals by plants.71 Adding high amounts of strong chelators such
as EDTA (ethylenediaminetetraacetate) to soils causes sorbed metals
to be released from soil and metal chelates to be formed, making the
metal mobile. In order for added chelating agents to be effective
in increasing metal uptake, huge amounts have to be applied to soil.
For example, induced phytoextraction of Pb required the addition of
10 mmol of EDTA kg–1 of soil which would cost over
$30 000 ha–1.71 EDTA was only effective when, after binding other metals in the
soil, there was some free EDTA which attached to the root membranes,
causing them to become leaky. High uptake of PbEDTA kills plants quickly.
The added EDTA, however, also causes metal leaching, and use of EDTA
to induce phytoextraction of soil metals is not allowed in the open
environment.72 Occasionally excessive fertilizer
rates of FeEDTA or ZnEDTA cause phytotoxicity in the field or greenhouse.

Our experience with metal chelation in soils and metal chelate
injury of plants provides insight into whether glyphosate would be
expected to affect plant uptake of micronutrient cations. If low concentrations
of EDTA or similar strong chelators are added to soils, they can promote
uptake of strongly adsorbed metals because the dissolved metal-chelate
can move metals from soil particles to the root membranes, circumventing
the diffusion limitations of metal uptake. Thus, in general, addition
of kg per ha levels of glyphosate might be expected to increase element
uptake if glyphosate were a strong chelator. However, glyphosate is
a relatively weak metal cation chelator compared to EDTA73−75 (Table 1). In general, none of the research
on chelating agent effects on metal uptake would indicate that a weak
chelator such as glyphosate would reduce or increase uptake of micronutrient
cations from soil.

Ratio of Glyphosate to Mineral Content in Glyphosate-Treated
Plants

Examination of glyphosate levels in glyphosate-treated
GR soybean seeds at maturity77 and mineral
levels in soybean seed78 shows that on
a molar basis the metal:glyphosate ratio can be from almost 10 000
times more Mn to around 100 000 times more minerals such as
Mg or Ca compared to glyphosate. Comparing glyphosate content of leaves
of glyphosate-treated GR soybean79 with
recently measured mineral content of GR soybean leaves,80 the ratios are smaller (ca. 300 for Ca, 30 for
Fe, 20 for Mn, and only 2 for Cu), but the ratio of total metal atoms
to glyphosate molecules is close to 1000. Even if a substantial fraction
of the minerals in the plant tissue were unavailable to glyphosate
due to chelation with other compounds, sequestration, or other means,
the ratio of mineral cations to glyphosate anions would still be very
large. These large ratios do not support the view that the chelator
properties of glyphosate would interfere substantially with plant
mineral nutrition in planta. Furthermore, at very
high in vivo concentrations of glyphosate in the
plant phloem, glyphosate has been calculated to be unable to effectively
compete for Fe–2+, Fe–3+, Ca–2+, Mn–2+, Mg–2+, Cu–2+, and Zn–2+ with biological
chelating agents.81

Effects of Glyphosate on Mineral Content of Soils

Soil
mineral status can affect plant mineral status. Reduction in soil
Mn concentrations due to glyphosate use has not been demonstrated.
In practice, glyphosate which reaches soil is strongly adsorbed by
Fe and Mn oxides and organic matter.27 When
glyphosate is bound by soil, it can be abiotically degraded82 in addition to the biodegradation pathways discussed
earlier. Other studies tested whether glyphosate reaching soil would
cause leaching of soil metals. Barrett and McBride83 tested leaching of metals in response to glyphosate application
for several soils and found that leaching occurred only with soils
highly contaminated with metals and only with high rates or repeated
applications of glyphosate. This outcome is predictable from the weak
chelation of metal ions by glyphosate. In contrast with some descriptions
of glyphosate as a strong chelator, the stability constants of glyphosate,
EDTA, and citric acid with common micronutrient ions show that glyphosate
is a weak chelator73−75 (Table 1). The fact that the
relative concentrations of metal cations in soil are several orders
of magnitude greater (in terms of moles of metals per ha vs moles
of glyphosate per ha) than the highest concentrations of glyphosate
that could be expected (discussed in detail in a previous section),
significant effects of glyphosate on soil mineral content or availability
to plants are highly unlikely.

Effects of Mineral Ions on Glyphosate Efficacy

From
the earliest days of glyphosate use, it was known that using water
containing high levels of metal ions would significantly reduce the
efficacy of the herbicide, presumably because the precipitated or
chelated herbicide is not taken up by target plants as well as the
free glyphosate anion and/or precipitation of glyphosate:mineral complexes
(reviewed by Duke,22 Sundaram and Sundaram,33 and Nilsson84). If
metal cations are present in a tank mix solution, and pH is raised
by addition of microelement fertilizer or by hard water, precipitation
of glyphosate reduces the plant uptake of glyphosate, thereby significantly
reducing its herbicidal effectiveness. The solubility of 1:1 metal/glyphosate
complexes decreases in the order of Mg ≈ Ca > Mn > Zn
> Cu
> Fe.33 In a 3 year study, Chahal et
al.85 found Ca, Mn, and Zn ions to reduce
glyphosate
efficacy on a variety of weeds when included in a tank mixture. Several
researchers have shown that separate application of Mn fertilizer
and glyphosate caused no effect or interaction, and recommend careful
consideration of tank mixes.86−88 EDTA, being a stronger chelator
than glyphosate, reverses the reduction of glyphosate herbicidal efficacy
by metal cations in spray tanks.89 Farmers
have been advised to not spray glyphosate with micronutrient plant
nutrition supplements unless the metal is chelated with a strong chelator
such as EDTA or EDDHA. Thus, studies on effects of glyphosate on mineral
nutrition of plants should not be conducted with combined spray solutions
of minerals and glyphosate. In short, a finding of metal ion precipitation
of glyphosate in a tank mix is not relevant to questions raised about
chemical interactions between glyphosate and micronutrients in plants
or soils.

Glyphosate Effects on Mineral Nutrition in GS Plants

Because glyphosate is a metal ion chelator, there was speculation
decades ago that this might be related to the mode of action of the
herbicide. However, the finding that GR crops with only a change in
their EPSPS are about 50-fold less sensitive to glyphosate than similar
GS crops79 indicated that mineral nutrition
is not involved in the mode of action of glyphosate. Further evidence
of this is the recent evolution of GR Palmer amaranth (Amaranthus
palmeri) biotypes that have multiple copies of the GS EPSPS
gene.90,91 The greater the number of copies of the
gene, the more resistant these plants are. If chelating Mn or any
other mineral was significantly involved in the mode of action of
glyphosate, this would not be the case.

Glyphosate can impede
absorption and translocation of calcium and magnesium in GS plants
(reviewed in Duke22). Nilsson84 found glyphosate to stimulate the accumulation
of Fe3+ in GS plants, while impeding movement of Zn2+ to the same sites. This result supports the finding that
subtoxic levels of glyphosate stimulate growth of iron-deficient wheat.84 Nilsson84 found no
effects of glyphosate on Mn, Zn, or Cu content of GS wheat leaves.
Eker et al.92 reported that glyphosate
reduced uptake and translocation of Mn and Fe in GS sunflower. Likewise,
Tesfamariam et al.93 found reduced Mn in
GS sunflower treated with glyphosate. Foliar-applied glyphosate to
GS soybean seedlings reduced uptake and translocation of Mg2+ and Ca2+, reduced tissue Ca content, and altered cellular
Ca distribution.94 Cakmak et al.95 found reduced levels of Ca, Mn, Mg, and Fe in
seeds and leaves of glyphosate-treated, GS soybean. In studies with
GS Festuca spp., Ca, Mg, Mn, and Fe were most reduced
by glyphosate treatment compared to other minerals.96 Such effects are readily explained by the known effect
of glyphosate on root growth and function in GS plants. Glyphosate
from foliar sprays is rapidly translocated to roots, where it strongly
inhibits root growth and other processes. Mineral uptake is highly
dependent on physiological regulation by growing young roots. Nearly
all of the multivalent metal cations are absorbed for translocation
to shoots by young roots.97−99

Bellaloui et al.20 reported reductions
in plant shoot Fe due to glyphosate application, resulting in chlorosis
in both GR and GS soybean cultivars. The authors correlated the effects
on Fe content with effects on root ferric reductase activity, however,
the methods used for measuring ferric reductase activity were inappropriate.
Roots grown in soil were removed, washed, and used in a bioassay of
FeEDTA reduction. Broken roots, loss of fine roots and root hairs,
and the presence of soil in the assay mixture confounds the measurement.
Ozturk et al.100 found inhibitory effects
of glyphosate on root ferric reductase in iron-deficient GS sunflower.
However, no in vitro effect of the herbicide on the
enzyme was reported to determine whether it was a primary or secondary
effect.

High rates of phosphate fertilizer have been reported
to remobilize
small amounts of glyphosate bound to soil.101 These low soil solution concentrations of glyphosate were phytotoxic
to a GS soybean cultivar on most soil types, but stimulated plant
growth (hormesis) on one soil type. Hormesis (the stimulatory effect
of a toxin at subtoxic concentrations) at low glyphosate doses is
a well-established phenomenon (e.g., Velini et al.102). However, the Bott et al.101 experiment has no relevance to practical field environments, as
the researchers applied extreme rates of dissolved superphosphate
to the surface of glyphosate-amended soils and planted the seeds immediately.
Fertilizer P rates are usually applied in bands below and to the side
of seeds to prevent adverse effects on seed germination. Considering
that the 240 mg P kg–1 highest rate of P application
would cause the amount in the surface 1–2 cm of the potted
soil to be 10–20 times higher than normally found in field
applications of P, one should not extrapolate from the results with
the high rates used in this study. It is questionable even whether
the low rates, where no adverse effects were observed, are relevant
to understanding glyphosate in the environment.

The studies
discussed in this subsection were done on GS plants,
so separating secondary effects of inhibition of EPSPS and effects
via any other mechanism is impossible. It may be that some of the
confusion regarding glyphosate effects on mineral nutrition of GR
crops is due to studies on GS plants that cannot be extrapolated to
GR plants.

The Cause of “Yellow Flash” Symptoms in GR Plants

As mentioned above, GR crops are highly resistant to glyphosate,
with resistance factors (I50 ratios between GR and susceptible
crops) of about 50 for both GR canola and GR soybean.79 No effects on growth of GR crops are normally seen at the
highest recommended field rates of glyphosate. Under some environmental
conditions with some cultivars, transient “yellow flash”
symptoms in GR soybeans are seen 5 to 20 days after glyphosate application
(Figure 5). Yellow flash has been attributed
to the rapid metabolism of glyphosate to the weakly phytotoxic AMPA
and not to mineral nutrition effects.103−108 GR crops are not necessarily resistant to AMPA, as its mode of action
is not the same as that of glyphosate. The yellow flash effect is
temporary and does not reduce yields, nor have yellow flash symptoms
been shown to be due to disease incidence in soybean. This yellowing
and interveinal chlorosis of rapidly growing young leaves in soybeans
experiencing yellow flash could be confused with symptoms of Fe or
Mn deficiencies. However, yellow flash symptoms are not accompanied
by effects on Mn status of the plant or on Mn uptake or distribution
by the plants.109 Yellow flash symptoms
have not been reported in GR crops other than soybean, perhaps because
sufficient levels of AMPA to cause such symptoms do not accumulate
in other GR crops or there is insufficient sensitivity of these crops
to AMPA. Little is known of AMPA in GR crops, including its mechanism
of phytotoxicity.41

Effects of Glyphosate on Mn in GR Crops

Huber110 suggested that use of glyphosate in production
of GR soybean leads to Mn deficiencies by reduction of Mn uptake and/or
translocation efficiency, changing soil/rhizosphere microbiology,
or modifying the form or availability of Mn in the environment. Dodds
et al.111,112 noted that GR-soybean cultivars showed lower
yield, stronger yellowing symptoms, and lower foliar Mn on a Mn marginal
or deficient soil than two conventional cultivars (non isolines).
Application of microelements had no effect on either soybean type.
It now appears that they observed that the GR-cultivar was inherently
less able to obtain soil Mn than the conventional cultivars.113 Mn deficiency can occur in soybeans grown on
low Mn soils such as the Lake Plain soils in the Midwest, and the
Coastal Plain soils on the east coast of the United States. If these
soils are limed, Mn becomes much less phytoavailable and soybeans
may suffer severe chlorosis and yield reduction until foliar Mn sprays
are applied or soil pH is lowered.114−116 Genetic variation for
susceptibility to Mn deficiency exists in soybeans (e.g., Graham et
al.115). Soybean cultivars for areas with
low phytoavailable soil Mn have been developed, and farmers are advised
to plant more Mn deficiency-resistant cultivars on such soils. As
breeders worked to solve this susceptibility problem (much like the
case of Fe chlorosis susceptibility of the early GR soybean cultivars;
see below), improved cultivars with the GR trait were also resistant
to Mn deficiency. This genetic variation in resistance to Mn deficiency
among soybeans occurs because roots change the microenvironment in
their rhizosphere to reduce Mn oxides to the soluble Mn2+, or reduce chelated Mn3+ with fulvic acids to promote
uptake by the roots. Local acidification of the rhizosphere may also
improve Mn uptake by cultivars resistant to Mn deficiency. Plants
also up-regulate metal ion transporters in their young roots to better
absorb the free Mn2+ in the rhizosphere.

Experiments
have been conducted in the field at multiple locations over multiple
years which found that there was no appreciable susceptibility to
Mn deficiency or need for Mn fertilizer to grow GR-soybean cultivars.36,117 Several field trials have shown that GR-soybeans are not commonly
experiencing Mn deficiency.80,86,103,113,117 Unfortunately, no study has been reported on soils which caused
clear Mn deficiency in soybeans in the absence of glyphosate so that
any interaction with glyphosate use could be measured.

There
are several peer-reviewed journal claims of effects of glyphosate
on mineral nutrition in GR soybean. Bott et al.10 reported that in the absence of glyphosate, a hydroponically
grown GR soybean cultivar accumulated more Mn than did a GS cultivar,
but the two lines were not near isogenic, making interpretation of
the data impossible. In addition, when both types of soybean were
grown with low Mn supply, there was no effect of glyphosate on shoot
concentration of Mn or growth. At very high application rates of glyphosate,
Mn concentrations in the tissue of the GR cultivar were reduced about
50%. There were no effects of glyphosate on Mn and Fe content of plant
tissues when the plants were grown in two different soil types, although
there was a reduction in insoluble foliar Zn in one of the soil types.
This tests whether the low molecular weight soluble chelates were
formed in the tissues as occurs with excessive EDTA. Taken together,
the data of this study show no adverse effect of glyphosate on Mn
uptake or translocation in GR-soybeans. Zobiole et al.12,14,15,17,19 reported that glyphosate treatment reduced
essential minerals (Mg, Mn, etc.) in GR soybean tissues. They also
reported dramatic reductions in photosynthesis associated with these
reductions,12,19 a result that is difficult to
reconcile with the high and increasing yields of these crops (see
section on yields below). In a more extensive study, Cavalieri et
al.118 examined the effects of 0.96 kg
ha–1 glyphosate from six different commercial formulations
on N, P, K, S, B, Ca, Mn, Mg, Fe, Zn, and Cu in two GR soybean cultivars
in the greenhouse. The results were equivocal, with both decreases
and increases in metals, depending on both cultivar and glyphosate
formulation. There was no clear pattern, other than reduced levels
of both metal and nonmetal elements as well as plant growth by one
formulation on one of the cultivars, suggesting that something other
than glyphosate was involved.

Comparing near-isolines of soybeans,
Loecker et al.117 found no effect of the
GR transgene of GR soybean
on Mn uptake or response to Mn in the absence of glyphosate. Rosolem
et al.107 found no effects of foliar application
of glyphosate on Mn absorption, accumulation, or distribution in GR
soybeans. Similar results were reported by Andrade and Rosolem.34 Serra et al.119 found
no effect of glyphosate doses up to 2.5 kg/ha on Cu, Mn, and Zn uptake
by GR soybeans, while Fe uptake increased at this high dose. No effects
of glyphosate on translocation of these metal ions were seen up to
2.5 kg/ha. In this study, exogenously applied Mn had no effect on
any responses to glyphosate. Lundry et al.120 found no effects of glyphosate on mineral nutrition in GR soybean
seeds, compared to an untreated near-isogenic soybean line, indicating
no effect from the EPSPS transgene or from glyphosate. Henry et al.36 found no glyphosate-induced deficiencies in
macronutrients (N, P, K, S, Mg, and Ca) or micronutrients (B, Zn,
Mn, Fe, Cu, and Al) in second generation GR soybeans. The application
of glyphosate to GR soybean had no effect on leaf mineral content
(Mn, Fe, Cu, and Zn) or yield at two different sites in Brazil.121 There was also no effect of absorption of exogenously
applied Mn. Exogenous Mn application had no effect on yield of glyphosate-treated,
GR soybeans, but it did enhance Mn and reduce Fe content in this study.
No effects 0.86 kg ha–1 glyphosate sprayed once
or twice on Mn content of both greenhouse- and field-grown GR soybean
leaves (young and old) or seed (Figure 6).80 There was no effect of glyphosate on yield in
this study. The results of all of these studies indicate that glyphosate
does not restrict the availability of micronutrients in glyphosate-treated,
GR crops. Thus, the results of the three research groups that have
reported glyphosate effects on mineral nutrition in GR crops are counter
to those of nine other research groups.

In many locations in IA, MN, ND, and some other
U.S. states, soybeans may suffer iron-deficiency-chlorosis (IDC) when
grown on wet calcareous soils.122−124 Soybean cultivars vary widely
in resistance to IDC, and many factors which influence soil moisture
and bicarbonate levels interact with severity of IDC.123,125 IDC can cause severe yield reduction on problem soils if cultivars
are not highly resistant to IDC.125−127 Because of the susceptibility
of many soybean cultivars to IDC, growers with problem soils are advised
to select chlorosis-resistant cultivars. Unfortunately, when GR soybeans
were first developed, the cultivars which were initially transformed
were not highly resistant to IDC, and many of the early high yielding
GR soybean cultivars developed for normal soils were susceptible to
IDC on wet calcareous soils. Soybean agronomists in states where IDC
is prevalent now screen genotypes for resistance to IDC and report
the results to growers so they can choose cultivars to match their
soil IDC problems. Thus, GR soybean cultivars have been screened for
susceptibility to IDC with and without glyphosate applications in
many locations. Although this has not been reported in the literature,
several scientists involved in soybean IDC screening confirm that
based on their observations in chlorosis rating field plots, glyphosate
causes no adverse interaction with iron deficiency in soybean (S.R.
Cianzio, Iowa State University; J.H. Orf, University of Minnesota;
T.C. Helms, North Dakota State University - Personal communications).

Possible Interaction of Glyphosate with Ni Phytoavailability

Although Duke et al.80 found no effect
of two applications of glyphosate on nickel content of leaves or seed
(Figure 6) of GR soybean, another report notes
that glyphosate use on GR-soybeans in a Brazilian study caused a significant
reduction in plant N-fixation and a decline in leaf Ni.14 Ni deficiency can reduce N-fixation. The foliar
Ni levels, even in their controls, were far below normal soybean foliar
Ni levels in other research. Ni is an essential element for all plants,128 but in the U.S. Ni deficiency of significant
consequence in the field has only been observed with some low Ni soils
of the southeastern Coastal Plain where pecans suffered severe deficiency
under some conditions of previous management which included raising
soil pH which reduces Ni phytoavailability.129 Legumes have a higher Ni requirement than nonlegumes because Ni
is needed for biochemical processes in nodule bacteria, as well as
for certain plant biochemistry. Unfortunately, Zobiole et al.14 did not test application of foliar Ni fertilizer
to confirm that the measured yield reduction actually resulted from
Ni deficiency induced by glyphosate. Furthermore, the level of Ni
in the Brazilian soil was not reported, so whether soil Ni deficiencies
were involved cannot be determined. That glyphosate is directly toxic
to some strains of Bradyrhizobium japonicum due the
fact that their EPSPS is also sensitive to glyphosate is well-known
(see section under Glyphosate Effects on Soil Microflora below), and this toxicity is not related to effects on Ni. Studies
designed to address the interactions of glyphosate and Ni metabolism
conducted on Ni-deficient soils with and without Ni supplementation
would be useful in interpreting the results of Zobiole et al.14

Mineral Content in Compositional Equivalence Studies in GR Crops

There are numerous studies on the compositional (chemical and nutritional)
equivalence of GR crops with GS crops, including mineral content,
although the intent of these papers was to evaluate the effect of
the transgene(s) on composition, rather than the effect of glyphosate
treatment on GR crops. In most of the published studies no mention
is made of whether glyphosate was used on the crop.130−132 In other studies the glyphosate application to the GR crop is not
completely described. For example, in Ridley et al.,133 the timing of glyphosate applications in GR corn is given,
but not the rates. In another study with GR corn134 the only information provided for the glyphosate treatments
was that they were made according to the label. A study with GR alfalfa
states only that glyphosate was applied prior to each cutting.135 More detailed information on the glyphosate
applications is provided in a study with GR corn by Ridley et al.136 For one set of trials, the GR corn received
an application of 1.08 kg ha–1, and in another ca.
0.85 kg ha–1 was used. The purpose of these studies
was to provide data required by regulatory agencies to determine the
effect of the transgenes on the composition of the harvested crop.
No effects of the GR transgenes or glyphosate application on mineral
content have been found in these field studies conducted under good
laboratory practices that usually involved multiple years and locations.
However, these studies lacked comparisons of glyphosate-treated with
untreated crops to allow evaluation of the glyphosate effect, independent
of the genetic effect of the GR technology.

Summary of Glyphosate Interactions with Plant Mircronutrient
Status

Clearly, glyphosate can have effects on mineral nutrition
of GS plants through its herbicidal effects on plant roots and other
parts of the plant. Published data on the effects of glyphosate on
mineral nutrition of GR crops are contradictory. Three groups have
claimed adverse effects on mineral nutrition in GR crops in peer-reviewed
journals—the Zobiole et al. group,12−15,17,19 Bellaloui et al.,20 and Bott et al.10 Others have made similar
claims in nonpeer-reviewed venues.111,112 The peer-reviewed
results of nine laboratories34,36,80,86,103,109,117−121 show no effect of glyphosate on mineral nutrition. These seemingly
contradictory results could be entirely or in part due to differences
in the soils, climatic conditions, and/or GR cultivars used. For example,
one group of experiments is based almost entirely on studies with
low pH soils using soybean varieties developed in Brazil and evaluated
in greenhouse studies.12−19 Rigorous field studies on different soil types (including those
highly susceptible to inducing Mn or Fe deficiency in soybeans) are
needed to resolve the issue of whether glyphosate might have adverse
effects on mineral nutrition of GR crops. Considering the available
data, growers are unlikely to need Mn fertilizers just because they
use glyphosate on GR soybeans.113

Glyphosate Effects on Soil Microflora

Soil microflora
can influence the persistence of glyphosate and
its metabolites in soil. Rhizosphere microflora can also influence
uptake of soil minerals by crops. Evaluation of glyphosate effects
on soil microorganisms requires knowledge of the direct effects of
glyphosate and its metabolites on soil microorganisms as well as effects
on microorganisms through processes mediated by plants on root symbionts
and rhizosphere microorganisms. The determination of relevant environmental
exposure concentrations needs to be compared to known response factors.
Finally, short-term and long-term responses on processes and community
structure need to be evaluated.

Glyphosate Toxicity to Microorganisms

As in plants,
glyphosate blocks the synthesis of the aromatic amino acids phenylalanine,
tyrosine, and tryptophan in some bacteria and fungi through the inhibition
of EPSPS, which also causes accumulation and excretion of shikimate-3-phosphate
and hydroxybenzoic acids in sensitive microorganisms.137,138 The sensitivity of bacterial EPSPS to glyphosate varies widely.
Pollegioni et al.42 divided microbial EPSPS
into two groups: sensitive (Class I) and relatively insensitive (Class
II). Class II includes Agrobacterium CP4 (the source
of the GR-EPSPS transgene in most GR-cultivars) in which the resistance
to glyphosate results from variations in the amino acid sequence of
EPSPS. Concentrations required for 50% inhibition were 75 μM
for E. coli, 174 μM for Bacillus subtilis, and 1100 μM for Pseudomonas aeruginosa EPSPS.137 Moorman et al.138,139 reported
variation in susceptibility of strains of Bradyrhizobium japonicum to glyphosate: 1000 μM (169 mg L–1) glyphosate
produced 47% inhibition for strain 110, but only 12 and 19% inhibition
for strains 123 and 138, respectively. Similarly, Hernandez et al.140 reported B. japonicum strains
ranging from sensitive to glyphosate (50% inhibition at 30 μM)
to insensitive (50% inhibition at >1000 μM). The full range
of resistance or sensitivity to glyphosate within the soil microbial
community is not fully known. Addition of aromatic amino acids to
bacterial cultures can partially or fully reverse the effects of glyphosate.
Some fungi are also sensitive to glyphosate, with 50% inhibition of
growth at concentrations of 5 to 50 mg/L (0.84–8.4 μM)
in culture.141

Understanding the
impact of glyphosate on soil microorganisms requires estimating concentrations
to which the microorganisms are exposed. Multiple applications of
glyphosate may occur in GR cropping systems. Glyphosate applied to
foliage is rapidly translocated to roots and other metaboically active
tissues.22 Glyphosate is exuded from roots
of treated plants into the rhizosphere,142−147 but the resulting concentrations in the rhizosphere soil are difficult
to document. Glyphosate applied to GS crops can be translocated to
the roots and released initially in exudates and later from decaying
tissues. As much as 15% of glyphosate applied to sensitive plants
could be translocated to roots.51,146 Similar patterns of
translocation were seen in GR-corn roots.148 Laitinen et al.146 also showed movement
of glyphosate from roots of treated plants to the soil, with the concentration
of glyphosate reaching 0.07 mg kg–1 soil in the
rhizosphere at four days after application.

Glyphosate may also
alter the quantity and quality of root exudates.
Kremer et al.145 compared carbohydrate
and amino acid exudation from roots of GR soybeans with or without
glyphosate treatment in hydroponic culture. Amino acid exudation was
increased by glyphosate, but carbohydrates (measured by an anthrone
reaction) were not different. Glyphosate treatment of a GS soybean
variety (Williams82) also resulted in increased
carbohydrate exudation. The root exudation of shikimate-3-P and protochatecuic
acid have not been examined, but exudation of these compounds might
be expected from GS plants after glyphosate application, as glyphosate
causes marked accumulation of these compounds in sensitive plants
(e.g., Lydon and Duke149).

Effects on Soil Microbial Populations and Community Structure

The effects of glyphosate on microorganisms in soil have been extensively
investigated using a variety of techniques. Two techniques that investigate
the community level responses, microbial biomass and respiration,
show either no effect or a temporary inhibition of respiration due
to glyphosate applied at rates less than 50 mg kg–1.150−153 At glyphosate application rates above 50 and up to 1500 mg kg–1 soil, soil respiration was stimulated. The range
of concentrations used in these studies resulted from different assumptions
about the penetration of sprayed glyphosate into the soil (see Lancaster
et al.151). The stimulatory effect of high
glyphosate concentrations on soil respiration is partly attributed
to microbial metabolism of glyphosate, but secondary effects due to
N and P mineralization could also stimulate respiration. These concentrations
of glyphosate seem sufficient to induce glyphosate toxicity; a hypothetical
application of 50 mg kg–1 glyphosate soil at 25%
gravimetric water content would result in a 1.18 mM aqueous concentration
in a thin layer at the surface of the soil. However, rapid adsorption
would reduce the concentration in the soil solution. A Kd of 50 would result in approximately 2% of the applied
herbicide being present in the soil solution resulting in an aqueous
concentration of approximately 24 μM glyphosate, which is sufficient
to affect sensitive microbial species. Community level measures, such
as respiration or total microbial biomass, are not sufficiently sensitive
to detect changes in population or activity of small subpopulations.

Alternatively, glyphosate impacts on soil microorganisms can be
assessed using measures of community structure and in long-term studies
where cumulative impacts may be determined. Hart and Brookes154 found no difference in microbial biomass, microbial
respiration and N mineralization in soils after 19 years of annual
glyphosate application compared to an untreated control soil. Busse
et al.155 compared Ponderosa pine (Pinus ponderosa) forest soils receiving glyphosate treatment
for understory vegetation control to control treatments (understory
cover at 25–100%). No glyphosate effects on soil respiration,
N mineralization, or microbial biomass were found when these plots
were evaluated after 9 to 13 years at each of the three sites. Powell
et al.156 compared a GR-soybean to a near
isoline sensitive cultivar over four years in Ontario. Rates of soybean
litter decomposition of the GR and conventional cultivars were nearly
identical; however, glyphosate reduced litter decomposition on the
soil surface, but not on buried litter. The ratios of fungal biomass
to bacterial biomass in the litter were only occasionally different,
with an increased ratio in the GR cultivars. Protists and nematode
populations were not affected.

Effects on Rhizosphere Populations and Community Structure

The rhizosphere is comprised of the root surface and the immediate
soil layer (2–5 mm) surrounding the root where microbial processes
are driven by root exudation of simple and complex substrates, which
include organic acids, flavonols, lignins, indole compounds, and amino
acids.157 The rhizosphere community includes
root symbionts, pathogens, plant growth-promoting rhizobacteria, phosphate-solublizing
bacteria, and microoganisms active in carbon and nitrogen cycling.158 Significant amounts of carbon are exuded from
growing roots, and rhizosphere populations may be exposed to glyphosate
through leaching of glyphosate from the soil surface and root exudation
of glyphosate.

Mijangos et al.159 examined glyphosate effects on GS plants (triticale and peas) and
their rhizosphere microbial communities. Ammonia concentrations increased
in rhizosphere soil after glyphosate treatment compared to the control
(no glyphosate, but clipped to remove above-ground biomass). Functional
diversity of the rhizosphere microbial community was examined using
a multiple substrate utilization test (Biolog Ecoplates) and genetic
diversity by denaturing gradient gel electrophoresis of 16S-rDNA after
PCR amplification. Community diversity and richness were reduced at
the highest rate of glyphosate application in rhizospheres of killed
GS pea and GS triticale, but not in soil from triticale grown alone.
The magnitude of these differences was similar to the differences
due to growing triticale alone or in combination with peas.

Several studies using different methods have examined the impact
of glyphosate on the rhizosphere of GR crops. Glyphosate application
to GR-soybean cultivars in the field in two growing seasons caused
transient differences in dehydrogenase activity, β-glucosaminidase
activity, β-glucosidase, and respiration.16 These enzyme activities are broadly distributed in soil
microorganisms, and the results suggest that broad spectrum toxicity
did not result from glyphosate application. Subsequent studies reported
increases in the ratio of Mn oxidizers/Mn reducers in response to
glyphosate and decreases in IAA-producing rhizobacteria.16 The magnitude of these responses increased as
the glyphosate application rate increased up to a rate equivalent
to 1.2 g ha–1. Manganese oxidation (Mn2+ + 1/2O2 + H2O→ MnO2 + 2H+) reduces the solubility of manganese. The observation that
glyphosate affects the ratio of Mn oxidizers/Mn reducers in the GR
soybean rhizosphere led to suggestions that glyphosate reduced plant
available Mn in soil and plant uptake of Mn.69 However, the extent that this shift to a higher ratio of Mn oxidizers
to Mn reducers has on the availability of Mn to plants was not determined.
Manganese is most available in soil under reduced conditions and/or
at low (<5.4) soil pH. A phylogenetically diverse group of both
bacteria and fungi are capable of Mn oxidation,160 but the cultural methods used to assess Mn oxidation or
reduction potential16 may not measure all
the microorganisms capable of Mn transformation, or their in situ activity. Also, plant roots actively regulate their
ability to obtain Mn from soils, up-regulate Mn transporters, and
secrete reducing materials which would release Mn from bound forms
in the soil for plant uptake. Additional research is needed to investigate
glyphosate-induced changes in Mn bioavailability in the rhizosphere.

Lupwayi et al.161 reported reduced functional
diversity (also using the multiple substrate utilization test) in
response to two glyphosate applications to GR-canola. Hart et al.162 found that rhizosphere populations of denitrifying
bacteria and fungi were not affected by glyphosate application to
GR-corn compared to GR-corn treated with conventional herbicides or
a GS corn isoline treated with conventional herbicides.

Barriuso
et al.163 extracted bacterial
DNA from GR corn rhizospheres after pre-emergence treatment with no
herbicide, glyphosate, or GTZ (a mixture of the herbicides acetochlor
and terbuthylazine). Pyrrosequencing of cloned 16S-rDNA showed that
microbial community structure after glyphosate treatment more resembled
the control (no herbicide) than the GTZ-treated community. Glyphosate
reduced Actinobacteria relative to the untreated control and Proteobacteria
were relatively unaffected. The GTZ treatment reduced microbial diversity
relative to the glyphosate or no-herbicide treatments. In contrast,
Lancaster et al.52 showed a variable response
of Actinobacteria populations to one or five applications of glyphosate
to soil without a crop, while Proteobacteria were increased by glyphosate
applications. The concentrations of microbial fatty acid methyl-esters
(FAME) from gram-negative bacteria also increased, which is consistent
with the increase in Proteobacteria populations.

Longer-term
(3 year) studies identified three microbial groups
dominating the GR corn rhizosphere in two fields in Spain: the Proteobacteria,
Actinobacteria, and Acidobacteria.164 Glyphosate
was applied postemergence to GR-corn, and roots were sampled 7 days
after glyphosate treatment and just prior to harvest. DNA extraction
and sequencing provided a database that was screened for 16S-rDNA
phylogenetic sequences. The abundance of these groups indicated little
effect of glyphosate over three years (Figure 7). Analysis of the same data with a clustering procedure showed that
the rhizosphere community was most affected by year and field and
least affected by time of sampling and herbicide. Acidobacteria increased
over time in both fields (Figure 7), while
Actinobacteria tended to decrease.

Lane et al.165 also
used FAME biomarkers
to examine the effects of two postemergent glyphosate applications
to GR-soybeans grown in soil with and without a history of previous
glyphosate use. At 7 days after application, total FAME (an indicator
of microbial biomass) was reduced in both soils. Nonmetric multidimensional
scaling of the FAME data showed a significant effect of the soil (history
vs no-history) on community structure, but no effect of application
or sampling times on community structure. The ratio of fungal to bacterial
biomass was also unaffected. The decrease in microbial biomass at
7 days after application does not support the conjecture that glyphosate
treatment increases root exudation. Weaver et al.54 also used FAME analysis to compare rhizosphere and bulk
soil community structures after glyphosate application to GR-soybean
in the field. After the second in-season glyphosate application, the
community structure of the bulk soil differed from that of the rhizosphere,
but two previous applications of glyphosate had no effect on FAME.
The same study included two fungal FAME biomarkers (16:1 ω5c
and 18:2 ω6c), and these were not affected by the glyphosate
treatments. The 16:1 ω5c (hexadecenoic acid) content is a biomarker
for arbuscular mycorrhizal fungi,166 while
18:2 ω6c is a more broad fungal marker, including Rhizoctonia
solani and Fusarium oxysporum.(167)

Nodulation and N-fixation

Zablotowicz and Reddy168 summarized the effects of glyphosate on soybean
nodulation and N fixation. GR soybeans treated with glyphosate had
reduced nodulation, as well as delayed N fixation, plant biomass accumulation,
and N fixation, but the severity of these effects was dependent upon
several factors. These included when glyphosate was applied to the
soybean, the number of glyphosate applications, the glyphosate formulation,
and the GR-soybean cultivar. Powell et al.169 compared nodule number and mass in six GR and three near isoline
GS soybean cultivars in the absence of glyphosate. Significant differences
in nodulation were found among the cultivars, but these were not related
to glyphosate resistance. Concentrations of glyphosate in nodules
and roots of soybeans were low (<200 ng g–1 nodule
tissue), although shikimate and hydroxybenzoic acids were present
in three-to 4-fold greater concentrations, indicating inhibition of B. japonicum EPSPS.168 Among
strains of B. japonicum, glyphosate tolerance in
culture was correlated with N fixation in excised nodules (acetylene
reduction assays).

Multiple field studies show no effect of
glyphosate on GR-soybean yield.170 Using
differences in natural abundance of 15N, Bohm et al.171 estimated the percentage of soybean N derived
from fixation to be 80% for GR soybean without glyphosate, 57% for
the same cultivar with one glyphosate application, and 66% after two
applications. Yield was not affected, and Bohm et al.171 suggested that the glyphosate-treated soybeans obtained
more reduced N from the soil. B. japonicum growth
in culture is reduced by glyphosate, but Rhizobium spp. degrade glyphosate when glyphosate toxicity is alleviated with
aromatic amino acids.44 These effects on
nodulation and N fixation may be due in part to the inhibitory effects
of glyphosate on B. japonicum, but may also be related
to GR cultivar responses to glyphosate. Additional evidence of cultivar
variability was found in a field study using 20 GR soybean cultivars
with and without glyphosate applied at four combinations of rates
and timings.172 Of the 20 cultivars, 9
showed no difference in nodule biomass compared to the unsprayed treatment.
Nodulation was reduced by as much as 61% for one cultivar. One GR
cultivar, BRS 244 RR, which had no glyphosate effect on nodule biomass
in this study, was reported to have reduced nodulation after glyphosate
application in a subsequent study.17 The
survival of B. japonicum in soil without plants was
not affected by concentrations equivalent to 1X or 10X field application
rates of glyphosate.139 Selection or construction
of GR B. japonicum would be an effective strategy
for alleviating negative effects on nodulation and N fixation.

The glyphosate metabolite AMPA can temporarily reduce chlorophyll
content (causing yellowing or chlorosis) and photosynthesis in GR
soybeans, particularly after foliar applications of AMPA at 1.0 kg
ha–1 or high rates of glyphosate.104,108 This rate was chosen to represent the complete metabolism of a glyphosate
application to AMPA. This rate of AMPA did not affect nodulation or
nitrogenase activity, suggesting that B. japonicum is less sensitive to AMPA than soybeans. The responses of GS cultivars
were similar to the GR cultivars, which are explained by the fact
that AMPA does not affect EPSPS. The mechanism of action of AMPA is
unknown.

Arbuscular Mycorrhizae

Arbuscular mycorrhizal fungi
are obligate symbionts that transfer mineral nutrients to their plant
hosts.173,174 Savin et al.175 evaluated glyphosate effects on arbuscular mycorrhizal fungi (AMF)
colonization of GR cultivars of cotton, corn and soybean grown in
soil under greenhouse conditions. AMF colonization of roots was not
affected by glyphosate, and neither were acid nor alkaline phosphatase
soil enzyme activities. Similar results were obtained by Knox et al.176 Other research has shown that in the tripartite
symbiosis of mycorrhiza, rhizobium, and soybean, no adverse effects
of glyphosate use on GR cultivars was observed.169 These studies indicate that effects of glyphosate on plant
mineral nutrition through effects on AMF are unlikely.

Effects of Glyphosate on Plant Disease in Glyphosate-Resistant
Crops

Plants use a variety of preformed and postinfection-induced
defenses
to resist pathogens. These include phenolic compounds which are considered
to be major components of defense across the plant kingdom.177−179 Phenolic compounds may act in defense as preformed antibiotics,
pathogen-induced phytoalexins, or as structural barriers in the form
of lignin. Thus, it is not surprising that any alteration in phenolic
metabolism may have an impact on the expression of disease. For example,
treatment with inhibitors of phenylalanine ammonia lyase have been
shown to enhance disease susceptibility (e.g., Holliday and Keen180), while treatment with compounds such as microbial
elicitors181 or even herbicides such as
the protoporphyrinogen oxidase (PPO) inhibitor lactofen can stimulate
accumulation of phenolic compounds and enhance disease resistance.182

Because glyphosate inhibits EPSPS, a
key enzyme in the shikimic
acid pathway, it also inhibits the biosynthesis of phenyalanine-derived
phenolic compounds and should also result in lack of synthesis of
salicylic acid from isochorismate.183 Hence,
this herbicide may enhance susceptibility to diseases in plants that
are susceptible to glyphosate. In addition to phenolic compounds,
glyphosate should also prevent synthesis of anthranilic acid, an intermediate
needed for the synthesis of the indole-based glucosinolates and phytoalexins
in crucifers184,185 and the avenalumin phytoanticipins186 and avenanthramide phytoalexins187 in oats. Finally, many plants respond to infection
by strengthening their cell walls with hydroxyproline-rich glycoproteins.188 Because these proteins contain a significant
amount of tyrosine, and formation of isodityrosine cross-links is
important in cell wall reinforcement, it would seem likely that glyphosate
may also impact this aspect of plant defense. However, the effect
of glyphosate on these nonphenylpropanoid based defenses has not been
reported. A summary of possible effects of glyphosate on plant defenses
derived from the shikimic acid pathway in shown in Figure 8. Based on both known and potential effects of glyphosate
on disease defense compounds, it is not surprising to find reports
in the published literature that show the disease-enhancing effects
of glyphosate on GS plants (e.g., reviewed in Johal and Huber69 and Duke et al.192).

Fungal and Oomycete Diseases

Research with GS Plants and Fungal Diseases

The effect
of glyphosate on disease resistance of GS plants was initially reported
by Keen and co-workers190 in 1982. They
reported that treatment of GS soybean hypocotyls with glyphosate decreased
the resistance to Phytophthora megsaperma and reduced
the accumulation of the isoflavonoid phytoalexin glyceollin. In a
subsequent study, Ward (193) confirmed the
results of Keen et al.190 and also showed
that glyphosate also reduced the efficacy of metalaxyl, a fungicide
specific for oomycetes.

The resistance breaking effect of glyphosate
has also been tested in two GS bean pathosystems. Pretreatment of
bean hypocotyls with glyphosate resulted in only a subset of the plants
becoming more susceptible to infection with an incompatible (avirulent)
race of Colletotrichum lindemuthianum.189 The increase in susceptibility was associated
with a decrease in phytoalexin production, but it is important to
note that the response was not uniform. A much different result was
found in the bean–Pythium interaction.194 In this case, treatment with glyphosate greatly
increased susceptibility of roots to Pythium. This
change in host reaction was associated with reduced accumulation of
phenolic phytoalexins and deposition of lignin.191

Although treatment of GS plants with glyphosate can
result in increased
susceptibility to pathogens, it is important to know if GR crops can
be predisposed to susceptibility by treatment with glyphosate. Biochemically,
it would seem unlikely that GR plants would become more susceptible
after glyphosate treatment, but is that the case? Johal and Huber69 and Kremer and Means195 reviewed glyphosate effects on GS and GR cultivars and suggested
that fungal root diseases were increased by the adoption of GR-cultivars
and the increased use of glyphosate.

GR Soybean and Sclerotinia Stem Rot (White Mold)

Several
studies have addressed whether or not GR plants are more susceptible
to disease, with much of the work focusing on GR soybeans. Lee et
al.196 tested two near-isogenic lines of
soybean GL2415 (GS) and GL2600RR (GR) for susceptibility to Sclerotinia sclerotiorum, the cause of the Sclerotinia stem
rot or white mold disease. Using a detached leaf assay, there was
no significant difference in lesion development in nontreated GL2415
as compared to GL2600RR. The formulation blank for the glyphosate
product used in the work also had no effect on disease. Most important
was the observation that treatment of GL2600RR with three different
rates of glyphosate did not increase the severity of disease in that
line as compared to the untreated controls for both GL2600RR and the
GS line GL2415. Thus, the conclusions for this work were that the
GR gene had no impact on disease and treatment of the GR plants with
glyphosate did not enhance susceptibility. Nelson et al.197 provided further evidence that the GR trait
did not impact host reaction to S. sclerotiorum in
field studies. Comparing four lines of soybean that were near-isogenic
for the GR trait, they found that, with one exception, the glyphosate
resistance trait had no effect on disease reaction. Glyphosate treatment
of two GR lines increased disease as compared to the untreated, while
the opposite effect was observed with two others. However, the two
GR lines that showed increased disease after glyphosate treatment
and their near-isogenic lines were significantly more susceptible
to Sclerotinia as compared to the two other lines
in which glyphosate had no effect on disease. Interestingly, treatment
of the two most white mold susceptible GR lines with another soybean
herbicide (thifensulfuron) resulted in enhanced disease development
comparable to the plants treated with glyphosate. Lactofen, a PPO
inhibitor known to induce resistance to S. sclerotiorum,182 was able to reduce disease severity
in all lines regardless of the presence or absence of the GR gene.
Perhaps most significant was the observation that glyphosate treatment
of GR lines had no effect on yield regardless of the amount of disease.

Lee et al.198 also addressed the issue
of cultivar differences in Sclerotinia stem rot susceptibility by
examining management options. This work further illustrated that issues
related to greater susceptibility of GR soybeans was not related to
glyphosate resistance trait, but rather to the susceptibility of the
cultivars that were used for transformation.

A lack of impact
on defense responses in GR soybean was supported
by analysis of glyceollin accumulation in resistant as compared to
susceptible plants.199 Using silver nitrate
as an elicitor, glyphosate treatment did not reduce the accumulation
of glyceollin.

GR Soybean, Fusarium, and Sudden Death Syndrome

Glyphosate application to some GR-soybean cultivars increases Fusarium spp. infection of roots in greenhouse experiments16,195 and under field conditions.195 For example, Fusarium spp. colonization increased from 20 to 30 infections
per 100 cm of untreated soybean root to as little as 30 infections
or as much as 120 infections per 100 cm root, depending upon glyphosate
dose and soybean growth stage and cultivar.16 In the same studies, decreases in populations of Pseudomonas spp. and indole acetic acid (IAA)-producing bacteria, as well as
a reduction in the ratio of Mn-reducing to Mn oxidizing microorganisms
were observed. Fluorescent Pseudomonas populations
in the GR-rhizosphere were decreased by glyphosate application and
negatively correlated with Fusarium root colonization.195 These Fusarium infections
of soybeans roots developed from soil-borne inoculum, and the species
distribution of Fusarium was not determined.

The mechanisms of these glyphosate-mediated increases in Fusarium root infection in GR soybeans are not established.
Results of studies on translocation of 14C-glyphosate from
treated leaves into roots and rhizospheres indicate that beneficial
microorganism-inhibiting glyphosate concentrations could occur. Kremer
et al.145 showed that root exudates in
general increased from glyphosate-treated plants grown in soil-free
conditions. However, these studies did not establish that these root
exudates specifically stimulated the growth of Fusarium spp. Still, glyphosate-mediated changes in quantity and quality
of root exudates into the rhizosphere has not been sufficiently evaluated
as an influence on plant disease.

The effects of glyphosate
and glyphosate resistance in relation
to sudden death syndrome (SDS), in soybean caused by Fusarium
virguliforme (formerly Fusarium solani f.
sp. glycines) has also been examined. Sanogo et al.200 reported on the effects of glyphosate and two
other soybean herbicides (lactofen and imazethapyr) on SDS response
in two GR soybean lines (Pioneer 9344 and Asgrow 3701) and one GS
line (BSR101). Two of the lines, Pioneer 9344 and BSR 101, are susceptible
to SDS while the other line was noted by the authors as having “above
average tolerance”. In growth chamber tests, the foliar symptom
severity of glyphosate-treated plants was no different than the untreated
control or plants treated with imazethapyr. Lactofen treatment decreased
SDS severity. In a greenhouse test, the severity of foliar and root
symptoms of SDS was increased by both glyphosate and imazethapyr (with
the exception of foliar severity in BSR 101 in which glyphosate treatment
resulted in no difference from the control). These results suggested
that the glyphosate resistance trait did not impact SDS response and
that all three lines reacted to infection by Fusarium in a similar manner after herbicide treatments.

Sanogo et
al.201 later reported on the
field reaction of the same three soybean lines to F. virguliforme and treatment with the same three herbicides plus acifluorfen. They
examined both foliar symptoms and frequency of Fusarium isolation from roots, and found no significant cultivar-herbicide
interaction. There was also a lower amount of disease in the more
resistant line regardless of herbicide type as compared to the two
susceptible lines, and treatment with glyphosate, acifluorfen and
imazethapyr all increased disease severity in susceptible lines as
compared to controls. Lactofen, in general, had no effect on disease
severity compared to the controls. The authors concluded that there
was no change in host resistance to SDS as a result of glyphosate
treatment.

The effects of glyphosate treatments on SDS were
examined in ten
GR soybean lines from a variety of maturity groups.202 In work similar that of Sanogo et al.,200,201 Njiti et al.202 reported no effect of
glyphosate treatment on yield, foliar symptoms, or root infection.
The overall conclusion from this study is that the host genotype,
and not glyphosate resistance or treatment with glyphosate, was the
most important factor in determining the reaction of a cultivar to
SDS.

Lévesque et al.203 found
that
glyphosate sprayed on mixed populations of weed species caused increased Fusarium spp. infection in some weed species, but not in
others. The number of colony-forming units of Fusarium spp. per gram of dried soil increased after application of glyphosate,
but GS crops (corn, pea, cucumber, and bean) subsequently grown on
in the field were not affected. Powell and Swanton204 concluded that there was insufficient evidence to prove
a link between glyphosate and plant diseases associated with Fusarium spp.

GR Soybean and Rhizoctonia solani

Harikrishnan and Yang et al.205 examined
the effect of glyphosate and other herbicides on reaction of BSR 101
(GS) and Pioneer 93B01 (GR) to Rhizoctonia solani. In greenhouse studies, the severity of Rhizoctonia infection in autoclaved soil was not increased by glyphosate in
Pioneer 93B01 compared to the inoculated control. In fact, statistically
similar levels of severity were also observed after imazethapyr treatment.
In nonautoclaved soil, glyphosate actually reduced the severity of Rhizoctonia as compared to plants that were inoculated but
not treated with a herbicide. In two years of field trials, glyphosate
treatment of Pioneer 9344 resulted in no difference in response to Rhizoctonia infection based on shoot dry weight, Rhizoctonia severity and plant stand.

Effect of GR Soybean in Rotation with Cereals

GR soybeans
are a rotation crop with cereals, and recent reports have suggested
that glyphosate treatment of soybeans increases the occurrence of
Fusarium head blight of wheat and barley.206,207 Because of these observations, Bérubé et al.208 tested the effects of tillage and glyphosate
treatment of GR soybean on Fusarium head blight development in a subsequent
planting of wheat and barley. There was no measurable effect of treating
GR soybeans with glyphosate on development of head blight and accumulation
of mycotoxins in barley or wheat.

Other GR Crops

In sugar beet, GR varieties were tested
for the effects of glyphosate treatment on expression of disease caused
by Rhizoctonia solani and Fusarium oxysporum f. sp. betae.209 Inoculation
with R. solani isolate R-1411 (AG-4) resulted in
comparable amounts of disease in both B4RR and H16 whether or not
they were treated with glyphosate or a surfactant. However, inoculation
with R. solani R-9 (AG-2-2) revealed that glyphosate
treatment resulted in increased disease in B4RR as compared to H16.
This suggests that glyphosate did have a negative effect on resistance
in B4RR. Inoculation with F. oxysporum isolate Fob13
resulted in increased disease in glyphosate-treated B4RR and H16 as
compared to nontreated controls. There was no effect of glyphosate
treatment on infection of the two sugar beet lines by F. oxysporum isolate F19. In a two year field study with GR sugar beet, Barnett
et al.210 reported that glyphosate had
no effect on expression of Rhizoctonia crown and root rot in four
GR lines (Hilleshög 9027RR, Hilleshög 9029RR, Hilleshög
9028RR and Crystal). They also reported that glyphosate treatments
did not impact efficacy of the fungicide azoxystrobin. Using field
and greenhouse evaluations, a follow-up study by Barnett et al.211 confirmed that glyphosate treatment of GR lines
had no effect on reaction to Rhizoctonia. Their recommendation to
growers was to use GR sugar beet varieties with the greatest amount
of Rhizoctonia resistance.

Two wheat lines
that were near-isogenic for glyphosate resistance were tested for
the effect of glyphosate on disease caused by Rhizoctonia
oryzae, R. solani, Pythium ultimum and Gaeumannomyces graminis var. tritici.212 The GR lines were not more susceptible
to any of these pathogens than the lines from which they were derived.
Furthermore, glyphosate application to the GR lines did not increase
disease severity. However, this study reported that volunteer GS wheat,
if killed by a foliar treatment with glyphosate resulted in increased
infection by R. solani and G. graminis var. tritici, possibly as a result of increased
amounts of pathogen inoculum produced in the crop residue.

The
reaction of GR cotton seedlings to Rhizoctonia solani after treatment with several pre- emergent herbicides and glyphosate
as a foliar treatment was tested in field and greenhouse experiments.213 Glyphosate applied at the cotyledon or four
leaf stage of GR cotton reduced Rhizoctonia infection of hypocotyls
in the field. In greenhouse studies, several pre-emergent herbicides
predisposed cotton seedlings to greater hypocotyl infection by R. solani, but subsequent application of glyphosate did
not increase severity of the disease. Baird et al.214 found that four varieties of GR cotton (PM 1220, DPL 5690,
DPL 5415, and DPL 50) had similar seedling stand count, height, and
dry weight when compared to GS varieties from the same lineage group,
regardless of glyphosate application. When differences did occur,
no consistent trends could be determined within the lineage groups
tested.

Glyphosate as a Plant Protectant

Glyphosate was shown
to have both preventive and curative activities against both stripe
rust (Puccinia striiformis f. sp. tritici)215,216 and leaf rust (Puccinia triticina) on GR wheat.215,217 In these cases, it appears that
glyphosate is acting directly as a fungicide. Some efficacy against Phakopsora pachyrhizi, the cause of Asian soybean rust,
was reported in both greenhouse215 and
in the field on GR soybeans.216 Tuffi Santos
at al.218 showed that glyphosate reduced
the severity of rust caused by Puccinia pdisii on Eucalyptus grandis. They found that there was a systemic
effect of glyphosate on rust development as illustrated by reduced
urediniospore germination and appressorium formation on tissues that
were not directly treated with the herbicide.

Similar to soybean,
glyphosate has recently been reported to protect GR alfalfa against
the rust Uromyces striatus when applied prior to
or up to 10 days after inoculation.219 In
this study, glyphosate was found to provide some protection against Colletotrichum trifolii and Phoma medicaginis. These latter two results are interesting as these pathogens, unlike
biotrophic rusts, are hemibiotrophic and necrotophic in their attack
of their hosts.

Bacterial Diseases

GS Soybean and Bacterial Blight

Holliday and Keen180 examined the effect of glyphosate on the response
of GS soybean leaves to the bacterial pathogen Pseudomonas
syringae pv glycinea. In this case, the
effect of glyphosate on resistance was less conclusive. Although glyphosate
treatment significantly decreased glyceollin accumulation, it had
no effect on the expression of the hypersensitive response. Glyphosate
treatment also resulted in only a relatively small increase in bacterial
growth in the treated plants. This suggests that in GS plants resistance
to bacterial blight is not greatly reduced after treatment with glyphosate.

GR Soybean and Bacterial Pustule

Several hundred GR
soybean lines were screened for resistance to bacterial pustule, caused
by Xanthomonas axonopodis pv glycines.220 The authors report that resistance
to the disease occurs in GR soybeans, but that not all genotypes were
resistant. Although they did not test the effect of glyphosate on
the host response to Xanthomonas, the authors did recommend that growers
assess the risk for this disease and plant resistant cultivars when
the disease is likely to occur.

GR Corn and Goss’s Wilt

Goss’s wilt and
leaf blight of corn, caused by the gram positive bacterium Clavibacter michiganensis subsp. nebraskensis, has increased over the last 5 years in corn-producing states,221−225 as has increased planting of GR corn (Figure 1). There are some logical explanations for the recent increase in
the occurrence of Goss’s wilt and leaf blight in corn growing
areas that do not implicate the use of GR corn or glyphosate. These
include continuous corn production over many years with minimal tillage.
Both of these practices will allow the buildup of pathogen inoculum
over time, and reduced tillage practices allow the pathogen to survive.
Reduced tillage practices that reduce residue decomposition will also
increase pathogen inoculum, although one of the perceived benefits
of GR crops has been the ability to manage weeds in reduced tillage.226,227 Another factor that may contribute to increased disease development
are reduced efforts to select for resistant hybrids and/or failure
to promote resistant hybrids by seed companies. Finally, weather events
(such as early season hail damage) will promote infection in even
the most resistant hybrids.221,228 Changes in C. michiganensis subsp. nebraskensis genotypes
might also be a factor, but further work is needed to determine if
this has occurred.222 There was no mention
in any of the recently published reports that the GR trait or glyphosate
application is a contributing factor to the increase in Goss’s
wilt. The report by Ruhl et al.225 noted
that the first Indiana finds were on both field corn and popcorn.
Since popcorn is not GR, this would further implicate other factors
in the recent outbreaks of the disease. Considering that most corn
produced in the US is now GR229 (Figure 1), it is likely that inoculum buildup and use of
GR corn that is not resistant to Goss’s wilt are the reasons
for increases in this disease. In addition, there are no reports in
the published literature that suggest that glyphosate resistance or
treatment of GR varieties with the herbicide will increase the risk
of other diseases in this crop.

Soybean Cyst Nematode

Yang et al.230 examined the effect of glyphosate on soybean cyst nematode
(SCN, Heterodora glycines) infection of the GR and
SCN-resistant variety Countrymark 316. Greenhouse tests demonstrated
no effect of glyphosate on SCN development on this genotype as compared
to untreated controls. Noel and Wax231 compared
the reactions of GR soybean lines DR 320 (SCN susceptible) and DSR
327 (SCR resistant) to glyphosate treatment and inoculation with H. glycines. They reported that glyphosate did result in
increased numbers of the nematode on the susceptible line, but not
the resistant line. Even with the increase in nematode populations,
there was no impact on yield. This study, like those with other soybean
diseases, suggests that genotypic resistance or susceptibility, rather
than glyphosate resistance, is the most important factor related to
disease severity.

Summary of Glyphosate and Disease Resistance

Although
it is clear that glyphosate does increase severity of disease on GS
plants, the published evidence for its effects on GR plants presents
a different story. Overall, it appears that in GR crops the baseline
disease resistance or susceptibility of the host plant, not the presence
of the glyphosate resistance gene or treatment with glyphosate, is
the major contributor to susceptibility.

Yields of Glyphosate-Resistant Crops

In the U.S., GR
soybeans, cotton, and corn were introduced in 1996,
1997, and 1998, respectively. Adoption of the crops has been rapid
and overwhelming, with more than 90% of soybeans, ca. 80% of cotton,
and about 70% of corn currently grown being GR (Figure 1). After the introduction of GR sugar beets in 2008, the adoption
rate was essentially 100% in 2009.

Thus, one might expect that
if there were any significant mineral
nutrition and/or disease problems with these crops, the problems would
be manifested in yield reductions and farmer dissatisfaction. Yield
data from the years before introduction of GR crops, continuing to
the present show that the same yield trends before introduction continued
after introduction (e.g., Figure 9). While
there could be isolated pockets of adverse effects of glyphosate on
GR crops that would be masked by their general success, such cases
have not been conclusively documented. There were initial concerns
with transgenic crops in general that there would be “yield
drags” due to factors not associated with disease or mineral
nutrition, but to suboptimal cultivars and potential pleiotrophic
effects of the transgenes.232 These problems
have not materialized. To summarize, yield data for crops that are
now predominantly GR cultivars do not support the view that there
are significant mineral nutrition or disease problems with GR crops.

Scientific accounts about increased plant disease
and mineral nutrition
problems in GR crops are based on publications from a limited number
of researchers. In the context of the entire body of relevant science,
the significance of these reports is questionable. Still, considering
the enormous importance of and reliance on GR crops and glyphosate,
there has been a paucity of publically funded research into potential
problems with this weed management technology. Farmers have generally
embraced this technology, so that there has been no widespread call
for studies of potential problems with GR crops other than those associated
with GR weeds, a growing problem that is well documented. Furthermore,
publication of negative (no effect) results is generally unattractive
to journals, and, therefore, to scientists whose success depends on
publications. So, the “no effect” papers that have been
published may not represent all such data that have been generated.
Reports of significant adverse effects of glyphosate on mineral nutrition
and diseases of GR crops are perplexing in light of the considerable
body of literature and yield data that contradict such claims. Nevertheless,
there might be effects of glyphosate in GR crops on mineral nutrition
and/or disease under particular but uncommon conditions (e.g., specific
soil, environmental conditions, particular GR crop cultivars, and/or
glyphosate formulations).

Notes

The authors
declare no competing financial interests.

Acknowledgments

We are
grateful to Theresa Varner for reviewing the USDA,
National Agricultural Statistics Service herbicide use and crop data
presented in the figures and Glenn Hanes for his technical assistance.
We gratefully acknowledge S. A. Ebelhar for providing Figure 5 from a field experiment that he conducted.

Krzysko-Lupicka T.,; Orlik A.,The use of glyphosate as the sole source of phosphorus or carbon
for the selection of soilborne fungal strains capable to degrade this
herbicide. ChemosphereYear: 1997, 34, 2601–2605.

Mamy L.,; Gabrielle B.,; Barriuso E.,Measurement and modelling of glyphosate
fate compared with that of herbicides replaced as a result of the
introduction of glyphosate-resistant oilseed rape. Pest Manag. Sci.Year: 2008, 64, 262–275.18205189

Robertson A.Will Goss’s
Wilt be the “Disease of the Year” in 2011. Iowa State University Extension (http://www.extension.iastate.edu/CropNews/2011/0805robertson.htm, accessed September 12, Year: 2012).

U.S. adoption of the three most widely grown,
herbicide-resistant
(HR) crops in the United States. Almost all HR crops during this period
were GR crops. Data for each crop category include varieties with
both HR as a single and stacked trait with insect resistance. Sources:
1996–1999 data are from Fernandez-Cornejo and McBride.1 Data for 2000–2012 are available in the
USDA, Economic Research Service data product, Adoption of
Genetically Engineered Crops in the U.S., Tables 1–3
(http://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-us.aspx, accessed September 12, 2012). Note that adoption data for 1996–1999
include HR corn and soybeans obtained using traditional breeding methods
(not transgenic). The more recent data (2000–2011) excluded
these varieties.

[Figure ID: fig2]

Figure 2

Agricultural herbicide usage in the U.S. Closed circles
= all herbicides
minus glyphosate, open circles = glyphosate only. Data from the U.S.
Department of Agriculture, National Agricultural Statistics Service
Data and Statistics site http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/ (accessed September 12, 2012).

[Figure ID: fig3]

Figure 3

Catabolic degradation
pathways of glyphosate.

[Figure ID: fig4]

Figure 4

Distribution of 14C after the (A) first or (B) fifth
glyphosate application to silt-loam soil The sequential glyphosate
applications were made at 2 week intervals. The extractable fraction
includes glyphosate and its transformation products extracted with
0.1 M NaOH.52.

[Figure ID: fig5]

Figure 5

Example of “yellow
flash” in GR soybeans sprayed
with glyphosate in Illinois.103

[Figure ID: fig6]

Figure 6

Effects of two, successive
glyphosate treatments (0.86 kg ai h–1 at both 3
and 6 weeks after planting) on the metal
content of mature seeds of field-grown GR soybean plants. Bars respresent
1 SE. There were no differences among any of the paired mean values
at the 95% confidence level.80.

[Figure ID: fig7]

Figure 7

Eubacterial phyla (16S-rDNA sequence abundance)
recovered from
GR-corn rhizosphere treated with glyphosate (G) or without glyphosate
(C) in two fields (upper and lower panels). Sampling was 7 days after
glyphosate application. Drawn from data from Barriuso et al.164

[Figure ID: fig8]

Figure 8

Possible effects of glyphosate treatment of glyphosate-senstive
plants on shikimic acid pathway metabolites considered to be important
in defense. Products of the shikimic acid pathway involved in plant
defenses are outlined by boxes. Metabolites and metabolic groups in
red have been demonstrated or are hypothesized to be reduced in GS
plants after glyphosate treatment. Of these, only isoflavonoid phytoalexins
from bean189 and soybean190 and lignin deposition191 in
bean have been examined for effects of glyphosate (and only in GS
plants). Those in black are expected to increase after glyphosate
treatment. Protocatechuic acid has been demonstrated to increase in
GS plant tissue after glyphosate treatment.149 **EPSPS: 5-enolpyruvylshikimic acid-3-phosphate synthase, the site
of glyphosate action.

[Figure ID: fig9]

Figure 9

U.S. yields
of the three crops over the past 30 years that are
now grown mostly as GR cultivars. The shaded area represent the years
since the introduction of each GR crop. Data are from the USDA, National
Agricultural Statistics Service Data and Statistics Web site: http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/ (accessed
September 12, 2012). GR crop adoption rates can be seen in Figure 1.

aAlthough protonated and deprotonated
chelates also occur, the 1:1 metal-ligand chelates are listed for
comparison using data from the Program Geochem-PC.70 Values for EDTA, citrate and glycine are for 0 ionic strength,
while the values for glyphosate are 0.1 M ionic strength.74 Values from AMPA are for 0.1 M ionic strength.76 Equilibria depend very strongly on solution
pH and the pKa values for the different proton binding functional
groups of a chelator molecule.